[en] The mechanism of ascorbate oxidation by metal-binding proteins (ceruloplasmin, albumin and transferrin) was investigated in vitro and in isolated plasma by the measurement of the ascorbyl free radicals (AFR) by electron spin resonance (ESR). In plasma of 13 healthy volunteers, a spontaneous and variable production of AFR was detected, which was increased by a 10(-4) M ascorbate overloading; however, this increase was not correlated to the intensity of the spontaneous AFR signal. The addition of Cu2+ and ceruloplasmin to plasma increased the ESR signal, while the addition of transferrin decreased the signal intensity in a dose-dependent manner. In vitro, we demonstrated that ascorbate was oxidized by human serum albumin and by ceruloplasmin, and that this oxidase-like activity was lost by trypsin or heat treatment of these proteins. These two proteins positively interacted in the oxidation of ascorbate, since addition of crude albumin to a solution of ascorbate and ceruloplasmin increased the intensity of ESR signal in a dose-dependent manner. The treatment of albumin by a metal chelator (DDTC) abolished these positive interactions. The respective roles of copper and iron in ascorbate oxidation were studied and showed a dose-dependent effect of these ions on ascorbate oxidation. The role of iron was confirmed by the inhibiting effect of metal-free transferrin on iron-dependent ascorbate oxidation. Concerted actions between iron carrying albumin and copper carrying ceruloplasmin appear responsible for the production of AFR in vitro and in vivo.
Research center :
CORD - Centre de l'Oxygène, Recherche et Développement - ULiège
Bielski BHJ, Comstock DA, Bowen RA. 1971 Ascorbic acid free radical. I. Pulse radiolysis study of optical absorption and kinetic properties. J Amer Chem Soc 93, 5624-5629.
Bielski BHJ. Allen AO, Schwarz HA. 1981 Mechanism of disproporlionation of ascorbate radicals. J Amer Chem Soc 103, 3516-3518.
Buettner GR, Jurkiewicz BA. 1993 Ascorbate free radical as a marker of oxidative stress: an EPR study. Free Radic Biol Med 14, 49-55.
Calabrese LC, Carbonaro M, Musci G. 1988 Chicken ceruloplasmin : evidence in support of a trinuclear cluster involving type 2 and 3 copper centers. J Biol Chem 263, 6480-6483.
Calabrese LC, Carbonaro M, Musci G. 1989 Presence of coupled trinuclear cluster in mammalian ceruloplasmin is essential for efficient electron transfer to oxygen. J Biol Chem 264, 6183-6187.
Curzon G, Young SN. 1972 The ascorbate oxidase activity of ceruloplasmin. Biochim Biophys Acta 268, 41-48.
Dodd NJ, Giron-Conland JM. 1975 Electron spin resonance study of changes during the development of a mouse myeloid leukaemia. II. The ascorbyl radical. Br J Cancer 32, 451-455.
Fox S, Karlin K. 1995 Dioxygen reactivity of copper proteins and complexes. In: Valentine JS, Foote CS, Greenberg A, Lieberman JF, eds. Active Oxygen in Biochemistry. London: Blackie Academic & Professional: 180-231.
Linder MC. 1991a Biochemistry of Copper. New York: Plenum Press; 242-262.
Linder MC. 1991b Biochemistry of Copper. New York: Plenum Press, 88-89.
Lohmann W. 1981 Ascorbate oxidase and its possible involvement in cancer. Z Naturforsch C 36, 804-808.
Lohmann W, Bensch KG, Muller E, Kang SO. 1981 ESR investigations on blood treated intravenously with ascorbic acid. Z Naturforsch C 36, 1-4.
Lovstad RA. 1995 A kinetic study of the coupled ironceruloplasmin catalyzed oxidation of ascorbate in the presence of albumin. BioMetals 8, 328-331.
Malmström BG. 1982 Enzymology of oxygen. Ann Rev Biochem 51, 21-59.
Martell AE. 1982 Chelates of ascorbic acid. Formation and properties. In: Seib PA, Tolbert BM, eds. Ascorbic Acid: Chemistry, Metabolism and Uses. Washington: American Chemical Society: 153-178.
Matsuo Y, Kihara T, Ikeda M, et al. 1995 Role of neutrophils in radical production during ischemia and reperfusion of the rat brain: effect of neutrophil depletion on extracellular ascorbyl radical formation. J Cereb Blood Flow Metab 1, 941-947.
Minakata K, Suzuki O, Saito S, Harada N. 1993 Ascorbate radical levels in human sera and rat plasma intoxicated with paraquat and diquat. Arch Toxicol 67, 126-130.
Minetti M, Forte T, Soriani M, et al. 1992 Iron-induced ascorbate oxidation in plasma as monitored by ascorbate free radical formation. No spin-trapping evidence for the hydroxyl radical in iron-overloaded plasma. Biochem J 282, 459-465.
Ortel TL, Takahashi N, Bauman RA, Putnam FW. 1983 The domaine structure of the human ceruloplasmin molecule. Protides Biol Fluids 31, 243-248.
Pietri S, Culcasi M, Stella L, Cozzone PJ. 1990 Ascorbyl free radical as a reliable indicator of free-radical-mediated myocardial ischemic and post-ischemic injury. A real-time continuous-flow ESR study. Eur J Biochem 13, 845-854.
Pietri S, Séguin JR, d'Arbigny PD, Culcasi M. 1994 Ascorbyl free radical: a noninvasive marker of oxidative stress in human open-heart surgery. Free Radic Biol Med 16, 523-528.
Roginsky VA, Stegmann HB. 1994 Ascorbyl radical as natural indicator of oxidative stress: quantitative regularities. Free Radic Biol Med 17, 93-103.
Sasaki, R, Kurokawa T, Tero-Kubota S. 1982 Nature of serum ascorbate radical and its quantitative estimation. Tohoku J Exp Med 136, 113-119.
Sasaki R, Kurokawa T, Shibua D. 1985 Factors influencing ascorbate free radical formation. Biochem Int 10, 155-163.
Sharma MK, Buettner GR, Spencer KT, Kerber RE. 1994 Ascorbyl free radical as a real-time marker of free radical generation in briefly ischemic and reperfused hearts. An electron paramagnetic resonance study. Circ Res 74, 650-658.
Vásquez-Vivar J, Santos AM, Junqueira VBC, Augusto O. 1996 Peroxynitrite-mediated formation of free radicals in human plasma: EPR detection of ascorbyl, albumin-thiyl and uric acid-derived free radicals. Biochem J 314, 869-876.
Yamazaki I, Piette L. 1961 Mechanism of free radical formation and disappearance during the ascorbic acid oxidase and peroxidase reactions. Biochim Biophys Acta 50, 62-69.
